More than 500 different protein kinases have been identified in humans and represent 1.7% of the human genome. Protein kinases function by catalyzing the transfer of the 3-phosphoryl group of ATP to the hydroxyl group on a specific protein substrate. The central role of kinases in disease and their fundamental role in human biology have prompted the development of potent and selective inhibitors as therapeutic agents and as novel tools for dissecting kinase function in the context of signal transduction pathways. Because many human kinases are so closely related both genetically and structurally, many inhibitors lack specificity for a single target and act on more than one kinase, typically by out-competing ATP in the active site. Hence there is a need for comprehensive kinase panels and tools for selectivity assessment of drugs. The kinase screening and profiling market was estimated to be $106 MM in 2008 and the demand is steadily rising every year. The purpose of our application is to develop a luminescence-based, homogeneous, non- radioactive, sensitive, cell-free assay that can be rapidly adapted for high throughput (HTP) screening and profiling of small molecule antagonists of kinases. The utility of this assay against commercially available drugs/dug candidates and the selectivity of these molecules against a kinase panel will be demonstrated. PUBLIC HEALTH RELEVANCE: Kinases constitute 1.7% of the genome and are implicated in various diseases, from cancer to diabetes. Assessment of selectivity of drugs against kinases is critical to evaluate their physiological safety. The purpose of our application is to develop a method to identify selective drugs by profiling them against a kinase panel.